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PCR/DNA Extraction Problem - (Jun/12/2010 )

I'm having an issue where I have been following the same methods for a few months now but recently my PCRs have started to produce smears with either no bands or bands amongst the smears.

Before (top right is positive control):


After (far right is positive control):


I am amplifying the ITS region for yeasts using the Chelex DNA extraction method. 96 samples are done in a PCR plate with the controls separately in 2 tubes (one pos one neg) with the same volume of master mix.

As you can see the positive control is working for the current gels without any streaking which I think indicates something with the DNA extraction but I have done quite a few now and I'm not sure that is the problem.

What can cause streaking on a gel which would only act on some samples and not the positive control?

I have tried to think of everything it could be but all the materials that have changed (different lots of ITS primers, new lot of loading dye, new lots of dNTP's) would all impact on the positive control as it is treated the same...

Happy to answer any further questions! :P

-_willo_-

I suspect that you might put too old colony or too much yeast cells into it. This is because when I see your gel, it is soooo bright. Over loading template will cause inhibitions of PCR, and over loading of cells will cause reduced effectiveness in removing nucleases or proteins by chelex solution. Try reduce the cells you put into chelex solutions and tell us whether you got any improvements.

Hope this helps
Adrian

-adrian kohsf-

adrian kohsf on Jun 14 2010, 10:28 AM said:

I suspect that you might put too old colony or too much yeast cells into it. This is because when I see your gel, it is soooo bright. Over loading template will cause inhibitions of PCR, and over loading of cells will cause reduced effectiveness in removing nucleases or proteins by chelex solution. Try reduce the cells you put into chelex solutions and tell us whether you got any improvements.

Hope this helps
Adrian


Hey, thanks for that. The brightness of the gel is due to different levels of exposure in the gel dock. However, I have asked others about my problem and they have also said it could be too many cells. When I put them in the eppendorfs for extraction (250uL 5% chelex solution) I tried to get as much as possible on a 20uL pipette tip so it is possible I simply put too much in. Am growing up some more from stocks now and re-extracting to see if there is a difference.

-_willo_-

Hi there, what I trying to say was the "well" of the gel...sorry for didn't mentioned earlier. This is because when i see most of the "loading well" which is "very bright", it seems that you do not get amplifications, especially from well 6 to well 11 from the second gel. Although some of the "bright" well still yield amplification, but i do still suspect you had loaded too much of template. try do a 100x dilution for the failed sample and run another PCR in the mean time you are growing your cells for your new DNA extraction.

Usually for most yeast (my experience), an overnight colony in broth will yield sufficient materials for DNA extraction. I even used to do a colony suspension from agar plate into sterile water and use it directly in PCR for ITS fungus identification (ITS1-ITS4 primers). But then, not all yeast works for my last method. Hope this helps.

Adrian

-adrian kohsf-

Well, I found what the cause of the problem was. After Adrian's reply and talking to some people in my lab I was sure I was simply loading too much DNA or it had too much 'junk' in it and just needed to dilute it. So I set up a small PCR and did some dilutions as well as a control with the normal 4uL I use for PCRs. The gel showed perfect bands for my control and fainter bands for the dilutions. Seeing as I used the same materials and methods I was stumped as to what could have caused the PCR to work perfectly. I then went and talked to some people and it turns out my problem was re-using the PCR plates. I thought washing them with ethanol and hot water and autoclaving them would get rid of anything inside but apparently not. After running the same extracts on brand new plates today I got perfect bands with no smearing.

Lesson learned: never re-use PCR plates!

-_willo_-

Re-use PCR plates??? .....LOL.....
^_^

-adrian kohsf-